PROJECT SUMMARY Ion channels are required to generate the electrical activity that drives contractility in the gastrointestinal tract. In the past grant cycle we have shown that human gastrointestinal interstitial cells of Cajal (ICC) express Ano1, a calcium-activated chloride (Cl-) channel belonging to the anoctamin family. We have also shown that Ano1 generates a current that due to alternative splicing has different electrophysiological properties in motility diseases such as gastroparesis. A central feature of the regulation of rhythmic smooth muscle contractile activity in the gastrointestinal tract is a Ca2+ signal that spreads from ICC to ICC in a highly coordinated fashion. The mechanism that underlies the synchronization of this Ca2+ signal is unknown. Understanding the mechanism is important as coordinated contractile activity is required for normal gastrointestinal function. The central hypothesis of this proposal is that Ano1 is required for synchronization of the ICC Ca2+ signal and subsequent coordinated smooth muscle contractility. We also hypothesize that we have identified the promoter region for Ano1 and that variants of Ano1 result in functional changes including the risk of developing delayed gastric emptying. We will test the central hypothesis in 2 specific aims. In SA1 we will determine the mechanisms by which Ano1 regulates coordinated gastrointestinal motility. In SA2 we will determine the regulation of Ano1 expression in health and disease including the promoter that drives Ano1 expression and the transcription factors involved. The SA are supported by preliminary data which show that absence of Ano1 expression is associated with loss of synchronization of the ICC Ca2+ signal, that knockdown of Ano1 in organotypic cultures disrupts Ca2+ signal synchronization, that smooth muscle contractility is uncoordinated when Ano1 expression is altered, that contrary to the published sequence for human Ano1, there is a completely missed exon that is 93 Kb away from the previously labeled exon 1 and that a polymorphism in this new exon is associated with gastroparesis. We also identified the presence of other anoctamins in ICC that when co-expressed with Ano1 results in cyclic electrical activity. An area proximal to the new exon 0 has properties suggesting it is the promoter for Ano1 and we identified relevant transcriptional factors including STAT6 and GLI1 that drive Ano1 transcription. We will use conditional knockout mice, Ca2+ imaging, spatiotemporal mapping, patch clamp techniques, microelectrode recordings, immunohistochemistry, Western blots, RNA seq, RT-PCR, single cell PCR, quantitative PCR, lentivirus RNA knock down techniques, and organotypic and single cell cultures to investigate the central hypothesis. The significance of this work is that, as a result of the work done in the previous grant cycles and the preliminary data presented in this proposal, we can significantly advance our understanding, at a cellular and molecular level, of the coordinated interaction between ICC and smooth muscle contractile activity and the contribution of Ano1 to normal and abnormal human physiology and risk of developing disease.